Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the roles of the core promoter and downstream elements in mediating regulation have been largely undefined. Basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. Basal transcription is completely dependent upon the general transcription factor TAF1 whereas activated transcription is TAF1 independent. We have also found that the downstream region of the MHC class I promoter region, between +1 and +32 bp, contains three novel regulatory elements. One of the elements,E+4, functions to increase transcription. The other two elements, DPE-L1 and DPE-L2 have sequence homology with previously characterized DPE elements, but are mechanistically and functionally distinct from other described DPE elements. Under constitutive conditions, the two DPE-L's act in concert to up-regulate promoter activity, preferentially increasing the use of TSS located at the 5' end of the cluster of multiple start sites. These elements promote constitutive, TAF1-dependent transcription. However, under activated TAF1-independent conditions, only one of the element functions independently as an enhancer. Thus, the downstream regulatory elements associated with the class I promoter function to fine-tune and integrate both intracellular and extracellular signaling pathways to ensure the appropriate level of MHC class I transcription to maintain immune homeostasis. These novel downstream elements are functionally and mechanistically distinct from previously described downstream elements in mammalian promoters. To further characterize regulation of class I gene expression, we have undertaken to characterize core promoter elements in vivo. To understand these mechanisms in more detail, we have characterized elements within the core promoter of the MHC class I gene. The minimal class I core promoter has been localized to a segment between -65 bp and +14 bp. Contained within this segment are a canonical CCAAT box, a TATAA-like element, an Sp1 binding site (Sp1BS) and an Initiator (Inr). In past studies, we have reported that no single element is necessary for expression of a reporter in transient transfection assays. However, we have now examined the dependence of transcription on each of the core promoter elements in the context of the native gene in transgenic mice. Remarkably, all of the mutant promoters supported transcription. Each element contributed uniquely to tissue-specificity, hormonal responses or both. The CCAAT box modulated constitutive expression in non-lymphoid tissues whereas the TATAA-like element controlled transcription in lymphoid tissues. The Sp1BS element and Inr negatively regulated constitutive transcription; the Inr regulated tissue specific patterns. Pol II binding, histone H3K4me3 patterns closely correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the WT, TATAA-like and CCAAT mutant promoters were activated by gamma-interferon, the Inr and Sp1 mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each contributes to fine tuning tissue-specific expression, extracellular signaling, promoter activity and chromatin structure. Transcription initiation is not a continuous process, but rather occurs in bursts which differ in amplitude and frequency. Although the roles of enhancers on the bursting characteristics of promoters in yeast and cultured mammalian cells have been described, the contributions of individual core promoter elements to bursting amplitude and frequency have not been assessed. Having demonstrated that the core promoter elements that constitute the MHC class I promoter all contribute to transcription initiation, we are examining their contribution to bursting characteristics in primary splenic B cells by single molecule RNA FISH.